Corneal endothelial cells are different from vascular and pulmonary xe2x80x9cendothelial cellsxe2x80x9d as they have a different embryonic tissue origin. Human corneal endothelial cells do not normally proliferate in vivo to replace those lost due to cell injury or death. Growth of these cells in culture is also extremely limited. This can be a serious problem as age, diseases such as glaucoma and diabetes, and ocular surgical procedures, such as laser vision correction and cataract extraction and intraocular lens implantation, cause an accelerated loss of these precious cells. There are no medical treatments for corneal diseases resulting from endothelial cell loss. Currently, corneal transplantation is the only way to restore normal vision.
The relative ability of corneal endothelial cells to proliferate in vivo and in culture appears to be a function of age; i.e., embryonic corneal endothelial cells and cells from neonates will proliferate much more readily than cells from children and adults. In a few cases, researchers have been able to culture cells from older donors, but growth has been supported by seeding the cells onto an artificial matrix, such as chondroitin sulfate/laminin, or onto extracellular matrix secreted by corneal endothelial cells from cows, one of a number of species whose corneal endothelial cells do grow readily in culture. A reliable way of supporting cell culture of human corneal endothelial cells would be highly desirable.
A new formulation for a culture medium that can be used to overcome such difficulties has now been developed. In general, the invention is directed to a formulation for a growth medium for culturing cells of neural crest origin, most preferably corneal endothelial cells, or for accelerating the growth and proliferation of such cells. The formulation for the nutrient medium of the invention includes nerve growth factor, preferably at a concentration of 1-100 ng/ml and most preferably 20 ng/ml. The growth medium formulation preferably also includes epidermal growth factor (preferably at a concentration of 1-200 ng/ml and most preferably 5 ng/ml). Most preferably, the formulation of the invention also includes fibroblast growth factor, at a concentration of 5-400 ng/ml or, most preferably, 40 ng/ml.
A commercial growth medium, particularly a serum-free medium, forms a useful basis for the growth medium of the invention. The growth medium of the invention can be augmented with serum, e.g., fetal bovine serum, as needed. As is well known to cell culture specialists, a useful human cell growth medium preferably also includes added ascorbic acid, human lipids, antibiotics and cell viability stabilizers.